The picornavirus enterovirus D-68 (EV-D68) is an icosahedral non-enveloped virus that was responsible for an outbreak of respiratory disease in the United States during 2014. During these outbreaks, acute flaccid paralysis similar to that caused by poliovirus was reported to correlate with EV-D68 infection in young children, suggesting that the virus might invade the central nervous system. EV-D68 is therefore an emerging human pathogen of medical significance. Like most other viruses, EV-D68 entry into the cell requires a receptor molecule. Two different cell receptors, sialic acid and decay-accelerating factor (DAF/CD55) have been reported to mediate EV-D68 entry. Replication of the NY68 and Fermon strains of EV-D68 in HeLa and A549 cells was unaffected by either neuraminidase treatment or the presence of antibodies against the SCR1 or SCR3 regions of DAF. However, infection by the Peru 68 strain in both cell lines was sensitive to treatment of cells with neuraminidase. These data suggest that more than one cell specific molecule is required for EV-D68 entry and that the molecule depends on the virus genotype. The goal of this proposal is to carry out a genetic screen for genes encoding molecules that participate in cell entry of EV-D68. To identify the cell receptor for EV-D68 we propose to genetically alter a population of cells using CRISPR-Cas9 methodology prior to infection with EV-D68, and identify those cells that no longer support viral replication. From this list of candidate genes, we will determine which molecule(s) functions during the early steps of viral replication: cell attachment, cell entry and viral uncoating using various tools including previous published work and bioinformatics. Cells determined to have disruptions in genes encoding transmembrane proteins and proteins with known characteristics to other picornavirus receptor proteins will be complemented by re-introducing the wild type gene into the corresponding altered cell. Cell binding and susceptibility to infection with multiple EV- D68 genotypes will be assessed by viral replication. If more than one cell protein is identified to mediate EV- D68 infection, we will refie our understanding of the virus-receptor interaction, by studying chimeric viruses in which the capsid coding region has been exchanged between the different genotypes.